Currently, five acyl-CoA dehydrogenase (ACDs) are known. These are short chain (SCAD), medium chain (MCAD), and long chain acyl-CoA dehydrogenases (LCAD), isovaleryl-CoA (IVD) and 2-methyl-branched chain acyl-CoA dehydrogenases (2-meBCAD). Electron transfer flavoprotein (ETF) serves as an electron acceptor for all ACDs. All ACDs are mitochondrial flavoproteins. They share a high degree of sequence similarity and belong to a gene family. Thus, their properties are similar, but ACDs discretely differ from each other in the chain length and configuration of their optimal substrate. Also, whereas LCAD, MCAD and SCAD catalyze the first reaction of the mitochondrial beta-oxidation of fatty acids, IVD and 2- meBCAD are involved in leucine and isoleucine/valine oxidation, respectively. Hence, the regulation of the first three may differ from those of the last two. The overall objectives of this proposal are (A) to characterize ACDs using site-directed mutagenesis techniques; (B) to search for the elements that regulate ACDs by studying the gene organization as well as by gene expression studies; and (C) to characterize several human ACD mutants using the site-directed mutagenesis and other molecular biological techniques. The individual projects are as follows: (1) Completion of the study of the structure of the IVD gene; (2) study of the organization of the SCAD and LCAD genes; (3) developmental and nutritional regulation of the tissue specific expression; (4) riboflavin-dependent expression of various ACDs; (5) structural basis of the substrate specificity: switching the chain length specificity of SCAD and LCAD; (6) structural basis of the substrate specificity: switching the substrate specificity of SCAD and IVD; (7) study of the catalytic and FAD-binding sties; (8) large scale preparation of normal human SCAD and IVD proteins for X-ray crystallographic study via prokaryotic expression; (9) characterization of the molecular defect of 304Glu-MCAD; (10) study of rare mutations of human MCAD; (11) the molecular and biochemical characterization of type II variant IVD; and (12) molecular characterization of human LCAD deficiency.